Antibodies to vascular endothelial cell growth factor

ABSTRACT

The present invention provides monoclonal antibodies, and portions thereof, which are capable of specifically binding to human vascular endothelial cell growth factor (hVEGF) or hVEGF-related protein. The invention also provides hybridoma cell lines that produce such monoclonal antibodies. The monoclonal antibodies of the invention are useful as therapeutic agents, either by themselves or in conjunction with cytotoxic or other chemotherapeutic agents, to treat diseases that are characterized by excessive vascular endothelial cell proliferation. The monoclonal antibodies of the invention also are useful in diagnostic and analytical methods for determining the presence of hVEGF or hVEGF related-protein in a test sample.

FIELD OF THE INVENTION

[0001] This application relates to hybrid cell lines (lymphocytehybridomas) for the production of monoclonal antibodies to humanvascular endothelial growth factor (hVEGF), to such homogeneousmonospecific antibodies, and to the use of such antibodies fordiagnostic and therapeutic purposes.

BACKGROUND OF THE INVENTION

[0002] The two major cellular components of the vasculature are theendothelial and smooth muscle cells. The endothelial cells form thelining of the inner surface of all blood vessels, and constitute anonthrombogenic interface between blood and tissue. In addition,endothelial cells are an important component for the development of newcapillaries and blood vessels. Thus, endothelial cells proliferateduring the neovascularization associated with tumor growth and a varietyof diseases, including psoriasis, arthritis, and diabetic retinopathy.

[0003] Various naturally occurring polypeptides reportedly induce theproliferation of endothelial cells. Among those polypeptides are thebasic and acidic fibroblast growth factors (FGF), Burgess and Maciag,Annual Rev. Biochem., 58:575 (1989), platelet-derived endothelial cellgrowth factor (PD-ECGF), Ishikawa, et al., Nature, 338:557 (1989), andvascular endothelial growth factor (VEGF), Leung, et al., Science246:1306 (1989); Ferrara & Henzel, Biochem. Biophys. Res. Commun.161:851 (1989); Tischer, et al., Biochem. Biophys. Res. Commun. 165:1198(1989); Ferrara, et al., PCT Pat. Pub. No. WO 90/13649 (published Nov.15, 1990); Ferrara, et al., U.S. Pat. App. No. 07/360,229.

[0004] VEGF was first identified in media conditioned by bovinepituitary follicular or folliculostellate cells. Biochemical analysesindicate that bovine VEGF is a dimeric protein with an apparentmolecular mass of approximately 45,000 Daltons, and with an apparentmitogenic specificity for vascular endothelial cells. DNA encodingbovine VEGF was isolated by screening a cDNA library prepared from suchcells, using oligonucleotides based on the amino-terminal amino acidsequence of the protein as hybridization probes.

[0005] Human VEGF was obtained by first screening a cDNA libraryprepared from human cells, using bovine VEGF cDNA as a hybridizationprobe. One cDNA identified thereby encodes a 165-amino acid proteinhaving greater than 95% homology to bovine VEGF, which protein isreferred to as human VEGF (hVEGF). The mitogenic activity of human VEGFwas confirmed by expressing the human VEGF cDNA in mammalian host cells.Media conditioned by cells transfected with the human VEGF cDNA promotedthe proliferation of capillary endothelial cells, whereas control cellsdid not. Leung, et al., Science 246:1306 (1989).

[0006] Several additional cDNAs were identified in the human cDNAlibrary that encode 121- and 189-amino acid hVEGF-related proteins. The121-amino acid protein differs from hVEGF by virtue of the deletion ofthe 44 amino acids between residues 116 and 159 in hVEGF. The 189-aminoacid protein differs from hVEGF by virtue of the insertion of 24 aminoacids at residue 116 in hVEGF, and apparently is identical to humanvascular permeability factor (hVPF). Keck, et al., Science 246:1309(1989); Connolly, et al., J. Biol. Chem. 264:20017 (1989); Keck, et al.,EPO Pat. Pub. No. 0 370 989 (published May 30, 1990).

[0007] In view of the fundamental role of vascular endothelial cellgrowth in many diseases, it is desirable to have a means of regulatingthe mitogenic effect of VEGF. It is also desirable to have a means ofassaying for the presence of VEGF in normal and pathological conditions,and especially cancer.

SUMMARY OF THE INVENTION

[0008] The present invention is directed to monoclonal antibodies, andportions thereof, which are capable of specifically binding to hVEGF orhVEGF-related protein. The invention also is directed to hybridoma celllines that produce such monoclonal antibodies. The monoclonal antibodiesof the invention are useful as therapeutic agents, either by themselvesor in conjunction with cytotoxic or other chemotherapeutic agents, totreat diseases that are characterized by excessive vascular endothelialcell proliferation. The monoclonal antibodies of the invention also areuseful in diagnostic and analytical assays for determining the presenceof hVEGF or hVEGF related-protein in a test sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows the effect of anti-hVEGF monoclonal antibodies(A4.6.1 or B2.6.2) or an irrelevant anti-hepatocyte growth factorantibody (anti-HGF) on the binding of the anti-hVEGF monoclonalantibodies to hVEGF.

[0010]FIG. 2 shows the effect of anti-hVEGF monoclonal antibodies(A4.6.1 or B2.6.2) or an irrelevant anti-HGF antibody on the biologicalactivity of hVEGF in cultures of bovine adrenal cortex capillaryendothelial (ACE) cells.

[0011]FIG. 3 shows the effect of anti-hVEGF monoclonal antibodies(A4.6.1, B2.6.2, or A2.6.1) on the binding of hVEGF to bovine ACE cells.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides monoclonal antibodies, or portionsthereof, which are capable of binding specifically to hVEGF orhVEGF-related protein. The term “hVEGF” refers to the 165-amino acidhuman vascular endothelial growth factor, and the term “hVEGF-relatedprotein” refers to the 121- and 189-amino acid proteins, that aredescribed by Leung, et al., Science 246:1306 (1989). The term“recombinant” used in reference to hVEGF or hVEGF-related protein refersto such proteins that are produced by recombinant DNA expression in ahost cell.

[0013] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts.

[0014] The monoclonal antibodies included within the scope of theinvention include hybrid and recombinant antibodies (e.g. “humanized”antibodies) regardless of species of origin or immunoglobulin class orsubclass designation, as well as antibody fragments (e.g., Fab, F(ab′)₂,and Fv), so long as they are capable of binding specifically to hVEGF orhVEGF-related protein. Cabilly, et al., U.S. Pat. No. 4,816,567; Mage &Lamoyi, in Monoclonal Antibody Production Techniques and Applications,pp.79-97 (Marcel Dekker, Inc., New York, 1987).

[0015] Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from such a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies of the invention may be made using the hybridomamethod first described by Kohler & Milstein, Nature 256:495 (1975), ormay be made by recombinant DNA methods. Cabilly, et al., U.S. Pat. No.4,816,567.

[0016] In the hybridoma method, a mouse or other appropriate host animalis immunized with hVEGF or hVEGF-related protein by subcutaneous,intraperitoneal, or intramuscular routes to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice,pp.59-103 (Academic Press, 1986).

[0017] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0018] Preferred myeloma cells are those that fuse efficiently, supportstable high. level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 cells available from the American Type CultureCollection, Rockville, Md. USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies. Kozbor, J. Immunol. 133:3001 (1984).Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63 (Marcel Dekker, Inc., New York, 1987).

[0019] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against hVEGF orhVEGF-related protein. Preferably, the binding specificity of monoclonalantibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).The monoclonal antibodies of the invention are those that preferentiallyimmunoprecipitate hVEGF or hVEGF-related protein that is present in atest sample, or that preferentially bind to hVEGF or hVEGF-relatedprotein in a binding assay.

[0020] In a preferred embodiment of the invention, the monoclonalantibody will have an affinity for binding hVEGF or hVEGF-relatedprotein of at least about 10 ⁹ liters/mole, as determined, for example,by the Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220(1980).

[0021] In another preferred embodiment of the invention, the monoclonalantibody is a neutralizing antibody. The term “neutralizing antibody” asused herein refers to a monoclonal antibody which is capable ofspecifically binding to hVEGF or hVEGF-related protein, and which iscapable of substantially inhibiting or eliminating the mitogenicactivity of one or more of such proteins with which it reacts. Typicallya neutralizing antibody will inhibit such mitogenic activity at leastabout 50%, and preferably greater than 80%, as determined, for example,by an in vitro cell survival or proliferation assay, such as describedin Example 2. The neutralizing antibodies of the invention areespecially useful in therapeutic applications, to prevent or treatunwanted endothelial cell proliferation or neovascularization.

[0022] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or neutralization activity, theclones may be subcloned by limiting dilution procedures and grown bystandard methods. Goding, Monoclonal Antibodies: Principles andPractice, pp.59-104 (Academic Press, 1986). Suitable culture media forthis purpose include, for example, Dulbecco's Modified Eagle's Medium orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal.

[0023] For some therapeutic and diagnostic applications, it is desirablethat the monoclonal antibody be reactive with fewer than all of thedifferent proteins within the group of hVEGF and hVEGF-related proteins.For example, it may be desirable to have a monoclonal antibody that iscapable of specifically binding to hVEGF but not to hVEGF-relatedproteins. Such antibodies are readily identified by comparative ELISAassays or comparative immunoprecipitation of hVEGF and each of thehVEGF-related proteins.

[0024] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0025] DNA encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells of the invention serve as a preferred source of suchDNA. Once isolated, the DNA may be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, ChineseHamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murinesequences, Morrison, et al., Proc. Nat. Acad. Sci. 81:6851 (1984), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Suchnon-immunoglobulin polypeptides include polypeptide toxins such asricin, diphtheria toxin, or Pseudomonas exotoxin (to produceimmunotoxins), viral sequences, cellular receptors such as a T-cellreceptor, cytokines such as TNF, interferons, or interleukins, and otherbiologically or immunologically active polypeptides. In that manner,“chimeric” or “hybrid” antibodies are prepared that have the bindingspecificity of an anti-hVEGF or anti-hVEGF-related protein monoclonalantibody.

[0026] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for hVEGF orhVEGF-related protein and another antigen-combining site havingspecificity for a different antigen.

[0027] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0028] For diagnostic applications, the antibodies of the inventiontypically will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g.,¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

[0029] Any method known in the art for separately conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter, et al., Nature 144:945 (1962); David, etal., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

[0030] The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,Inc., 1987).

[0031] Competitive binding assays rely on the ability of a labeledstandard (which may be hVEGF, hVEGF-related protein, an immunologicallyreactive portion thereof) to compete with the test sample analyte (hVEGFor hVEGF-related protein) for binding with a limited amount of antibody.The amount of hVEGF or hVEGF-related protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0032] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three part complex. David & Greene, U.S. Pat. No. 4,376,110.The second antibody may itself be labeled with a detectable moiety(direct sandwich assays), or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

[0033] The antibodies of the invention also are useful for in vivoimaging, wherein an antibody labeled with a detectable moiety isadministered to a host, preferably into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.This imaging technique is useful in the staging and treatment ofneoplasms. The antibody may be labeled with any moiety that isdetectable in a host, whether by nuclear magnetic resonance, radiology,or other detection means known in the art.

[0034] For therapeutic applications, the antibodies of the invention maybe administered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form, including those that may be administered to ahuman intravenously as a bolus or by continuous infusion over a periodof time, by intramuscular, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The antibody is alsosuitably administered by intratumoral, peritumoral, intralesional, orperilesional routes, to exert local as well as systemic therapeuticeffects.

[0035] Such dosage forms encompass pharmaceutically acceptable carriersthat are inherently nontoxic and nontherapeutic. Examples of suchcarriers include ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partialglyceride-mixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and polyethylene glycol. Carriers for topical or gel-basedforms of antibody include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol, and wood wax alcohols. For all administrations,conventional depot forms are suitably used. Such forms include, forexample, microcapsules, nano-capsules, liposomes, plasters, inhalationforms, nose sprays, and sublingual tablets. The antibody will typicallybe formulated in such vehicles at a concentration of about 0.1 mg/ml to100 mg/ml.

[0036] For the prevention or treatment of disease, the appropriatedosage of antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibodies are administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

[0037] Depending on the type and severity of the disease, about 0.015 to15 mg/kg of antibody is an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful.

[0038] According to another embodiment of the invention, theeffectiveness of the antibody in preventing or treating disease may beimproved by administering the antibody serially or in combination withanother agent that is effective for those purposes, such as anotherantibody directed against a different epitope or neutralizing adifferent protein than the first antibody, or one or more conventionaltherapeutic agents such as, for example, alkylating agents, folic acidantagonists, anti-metabolites of nucleic acid metabolism, antibiotics,pyrimidine analogs, 5-fluorouracil, purine nucleosides, amines, aminoacids, triazol nucleosides, corticosteroids, calcium, retinoids,lipoxygenase and cyclooxygenase inhibitors, fumaric acid and its salts,analgesics, psychopharmaceuticals, local anesthetics, spasmolytics, andbeta-blockers. Such other agents may be present in the composition beingadministered or may be administered separately. Also, the antibody issuitably administered serially or in combination with radiologicaltreatments, whether involving irradiation or administration ofradioactive substances.

[0039] The antibodies of the invention also are useful as affinitypurification agents. In this process, the antibodies against hVEGF orhVEGF-related protein are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing thehVEGF or hVEGF-related protein to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the hVEGF or hVEGF-relatedprotein, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent, such as glycine buffer,pH 5.0, that will release the hVEGF or hVEGF-related protein from theantibody.

[0040] The following examples are offered by way of illustration onlyand are not intended to limit the invention in any manner. All patentand literature references cited herein are expressly incorporated.

EXAMPLE 1 Preparation of Monoclonal Antibodies

[0041] To obtain hVEGF conjugated to keyhole limpet hemocyanin (KLH) forimmunization, recombinant hVEGF (165 amino acids), Leung, et al.,Science 246:1306 (1989), was mixed with KLH at a 4:1 ratio in thepresence of 0.05% glutaraldehyde and the mixture was incubated at roomtemperature for 3 hours with gentle stirring. The mixture then wasdialyzed against phosphate buffered saline (PBS) at 4° C. overnight.

[0042] Balb/c mice were immunized four times every two weeks byintraperitoneal injections with 5 μg of hVEGF conjugated to 20 μg ofKLH, and were boosted with the same dose of hVEGF conjugated to KLH fourdays prior to cell fusion.

[0043] Spleen cells from the immunized mice were fused with P3X63Ag8U.1myeloma cells, Yelton, et al., Curr. Top. Microbiol. Immunol. 81:1(1978), using 35% polyethylene glycol (PEG) as described. Yarmush, etal., Proc. Nat. Acad. Sci. 77:2899 (1980). Hybridomas were selected inHAT medium.

[0044] Supernatants from hybridoma cell cultures were screened foranti-hVEGF antibody production by an ELISA assay using hVEGF-coatedmicrotiter plates. Antibody that was bound to hVEGF in each of the wellswas determined using alkaline phosphatase-conjugated goat anti-mouse IgGimmunoglobulin and the chromogenic substrate p-nitrophenyl phosphate.Harlow & Lane, Antibodies: A Laboratory Manual, p.597 (Cold SpringHarbor Laboratory, 1988). Hybridoma cells thus determined to produceanti-hVEGF antibodies were subcloned by limiting dilution, and two ofthose clones, designated A4.6.1 and B2.6.2, were chosen for furtherstudies.

[0045] The A4.6.1 and B2.6.2 hybridoma cell lines have been depositedwith the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 USA, and are maintained by the ATCC under accessionnumbers HB 10709 and HB 10710, respectively.

EXAMPLE 2 Characterization of Monoclonal Antibodies

[0046] A. Antigen Specificity

[0047] The binding specificities of the anti-hVEGF monoclonal antibodiesproduced by the A4.6.1 and B2.6.2 hybridomas were determined by ELISA.The monoclonal antibodies were added to the wells of microtiter platesthat previously had been coated with hVEGF, FGF, HGF, or epidermalgrowth factor (EGF). Bound antibody was detected with peroxidaseconjugated goat anti-mouse IgG immunoglobulins. The results of thoseassays confirmed that the monoclonal antibodies produced by the A4.6.1and B2.6.2 hybridomas bind to hVEGF, but not detectably to those otherprotein growth factors.

[0048] B. Epitope Mapping

[0049] A competitive binding ELISA was used to determine whether themonoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas bindto the same or different epitopes (sites) within hVEGF. Kim, et al.,Infect. Immun. 57:944 (1989). Individual unlabeled anti-hVEGF monoclonalantibodies (A4.6.1 or B2.6.2) or irrelevant anti-HGF antibody (IgGlisotype) were added to the wells of microtiter plates that previouslyhad been coated with hVEGF. Biotinylated anti-hVEGF monoclonalantibodies (BIO-A4.6.1 or BIO-B2.6.2) were then added. The ratio ofbiotinylated antibody to unlabeled antibody was 1:1000. Binding of thebiotinylated antibodies was visualized by the addition ofavidin-conjugated peroxidase, followed by o-phenylenediaminedihydrochloride and hydrogen peroxide. The color reaction, indicatingthe amount of biotinylated antibody bound, was determined by measuringthe optical density (O.D) at 495 nm wavelength.

[0050] As shown in FIG. 1, in each case, the binding of the biotinylatedanti-hVEGF antibody was inhibited by the corresponding unlabeledantibody, but not by the other unlabeled anti-hVEGF antibody or theanti-HGF antibody. These results indicate that the monoclonal antibodiesproduced by the A4.6.1 and B2.6.2 hybridomas bind to different epitopeswithin hVEGF.

[0051] C. Isotyping

[0052] The isotypes of the anti-hVEGF monoclonal antibodies produced bythe A4.6.1 and B2.6.2 hybridomas were determined by ELISA. Samples ofculture medium (supernatant) in which each of the hybridomas was growingwere added to the wells of microtiter plates that had previously beencoated with hVEGF. The captured anti-hVEGF monoclonal antibodies wereincubated with different isotype-specific alkalinephosphatase-conjugated goat anti-mouse immunoglobulins, and the bindingof the conjugated antibodies to the anti-hVEGF monoclonal antibodies wasdetermined by the addition of p-nitrophenyl phosphate. The colorreaction was measured at 405 nm with an ELISA plate reader.

[0053] By that method, the isotype of the monoclonal antibodies producedby both the A4.6.1 and B2.6.2 hybridomas was determined to be IgG1.

[0054] D. Binding Affinity

[0055] The affinities of the anti-hVEGF monoclonal antibodies producedby the A4.6.1 and B2.6.2 hybridomas for hVEGF were determined by acompetitive binding assays. A predetermined sub-optimal concentration ofmonoclonal antibody was added to samples containing 20,000-40,000 cpm¹²⁵I-hVEGF (1-2 ng) and various known amounts of unlabeled hVEGF (1-1000ng). After 1 hour at room temperature, 100 μl of goat anti-mouse Igantisera (Pel-Freez, Rogers, AR USA) were added, and the mixtures wereincubated another hour at room temperature. Complexes of antibody andbound protein (immune complexes) were precipitated by the addition of500 μl of 6% polyethylene glycol (PEG, mol. wt. 8000) at 4° C., followedby centrifugation at 2000×G. for 20 min. at 4° C. The amount of¹²⁵I-hVEGF bound to the anti-hVEGF monoclonal antibody in each samplewas determined by counting the pelleted material in a gamma counter.

[0056] Affinity constants were calculated from the data by Scatchardanalysis. The affinity of the anti-hVEGF monoclonal antibody produced bythe A4.6.1 hybridoma was calculated to be 1.2×10⁹ liters/mole. Theaffinity of the anti-hVEGF monoclonal antibody produced by the B2.6.2hybridoma was calculated to be 2.5×10⁹ liters/mole.

[0057] E. Inhibition of hVEGF Mitogenic Activity

[0058] Bovine adrenal cortex capillary endothelial (ACE) cells, Ferrara,et al., Proc. Nat. Acad. Sci. 84:5773 (1987), were seeded at a densityof 10 ⁴ cells/ml in 12 multiwell plates, and 2.5 ng/ml hVEGF was addedto each well in the presence or absence of various concentrations of theanti-hVEGF monoclonal antibodies produced by the A4.6.1 or B2.6.2hybridomas, or an irrelevant anti-HGF monoclonal antibody. Afterculturing 5 days, the cells in each well were counted in a Coultercounter. As a control, ACE cells were cultured in the absence of addedhVEGF.

[0059] As shown in FIG. 2, both of the anti-hVEGF monoclonal antibodiesinhibited the ability of the added hVEGF to support the growth orsurvival of the bovine ACE cells. The monoclonal antibody produced bythe A4.6.1 hybridoma completely inhibited the mitogenic activity ofhVEGF, whereas the monoclonal antibody produced by the B2.6.2 hybridomaonly partially inhibited the mitogenic activity of hVEGF.

[0060] F. Inhibition of hVEGF Binding

[0061] Bovine ACE cells were seeded at a density of 2.5×10⁴ cells/0.5ml/well in 24 well microtiter plates in Dulbecco's Modified Eagle'sMedium (DMEM) containing 10% calf serum, 2 mM glutamine, and 1 ng/mlbasic fibroblast growth factor. After culturing overnight, the cellswere washed once in binding buffer (equal volumes of DMEM and F12 mediumplus 25 mM HEPES and 1% bovine serum albumin) at 4° C.

[0062] 12,000 cpm ¹²⁵I-hVEGF (approx. 5×10⁴ cpm/ng/ml) was preincubatedfor 30 minutes with 5 μg of the anti-hVEGF monoclonal antibody producedby the A4.6.1, B2.6.2, or A2.6.1 hybridoma (250 μl total volume), andthereafter the mixtures were added to the bovine ACE cells in themicrotiter plates. After incubating the cells for 3 hours at 4° C., thecells were washed 3 times with binding buffer at 4° C., solubilized bythe addition of 0.5 ml 0.2 N. NaOH, and counted in a gamma counter.

[0063] As shown in FIG. 3 (upper), the anti-hVEGF monoclonal antibodiesproduced by the A4.6.1 and B2.6.2 hybridomas inhibited the binding ofhVEGF to the bovine ACE cells. In contrast, the anti-hVEGF monoclonalantibody produced by the A2.6.1 hybridoma had no apparent effect on thebinding of hVEGF to the bovine ACE cells. Consistent with the resultsobtained in the cell proliferation assay described above, the monoclonalantibody produced by the A4.6.1 hybridoma inhibited the binding of hVEGFto a greater extent than the monoclonal antibody produced by the B2.6.2hybridoma.

[0064] As shown in FIG. 3 (lower), the monoclonal antibody produced bythe A4.6.1 hybridoma completely inhibited the binding of hVEGF to thebovine ACE cells at a 1:250 molar ratio of hVEGF to antibody.

[0065] G. Cross-Reactivity with VEGF-Related Proteins

[0066] To determine whether the anti-hVEGF monoclonal antibody producedby the A4.6.1 hybridoma is reactive with hVEGF-related protein, theantibody was assayed for its ability to immunoprecipate hVEGF-relatedprotein.

[0067] Human 293 cells were transfected with vectors comprising thenucleotide coding sequence of the 121- and 189-amino acid hVEGF-relatedproteins, as described. Leung, et al., Science 246:1306 (1989). Two daysafter transfection, the cells were transferred to medium lackingcysteine and methionine. The cells were incubated 30 minutes in thatmedium, then 100 μCi/ml of each ³⁵S-methionine and ³⁵S-cysteine wereadded to the medium, and the cells were incubated another two hours. Thelabeling was chased by transferring the cells to serum free medium andincubating three hours. The cell culture media were collected, and thecells were lysed by incubating for 30 minutes in lysis buffer (150 mMNaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50mM Tris, pH 8.0). Cell debris was removed from the lysates bycentrifugation at 200×G. for 30 minutes.

[0068] 500 μl samples of cell culture media and cell lysates wereincubated with 2 μl of A4.6.1 hybridoma antibody (2.4 mg/ml) for 1 hourat 4° C., and then were incubated with 5 μl of rabbit anti-mouse IgGimmunoglobulin for 1 hour at 4° C. Immune complexes of ³⁵S-labeledhVEGF-related protein and anti-hVEGF monoclonal antibody wereprecipitated with protein-A sepharose (Pharmacia), then subjected toSDS-12% polyacrylamide gel electrophoresis under reducing conditions.The gel was exposed to x-ray film for analysis of theimmunoprecipitated, radiolabeled proteins by autoradiography.

[0069] The results of that analysis indicated that the anti-hVEGFmonoclonal antibody produced by the A4.6.1 hybridoma was cross-reactivewith both the 121- and 189-amino acid hVEGF-related proteins.

What is claimed is:
 1. A monoclonal antibody capable of specificallybinding to hVEGF or hVEGF-related protein.
 2. A monoclonal antibody ofclaim 1 which inhibits the mitogenic activity of hVEGF or hVEGF-relatedprotein.
 3. A monoclonal antibody of claim 1 which has an affinity forhVEGF of at least 10⁹ liters/mole.
 4. A polypeptide comprising anantigen-combining site of an antibody of claim
 1. 5. An isolated DNAencoding an antibody of claim
 2. 6. A hybridoma cell which produces amonoclonal antibody of claim
 1. 7. A hybridoma cell which produces amonoclonal antibody of claim
 2. 8. A hybridoma cell which produces amonoclonal antibody of claim
 3. 9. The hybridoma cell deposited underAmerican Type Culture Collection Accession Number ATCC ______ (referredto herein as A4.6.1).
 10. The hybridoma cell deposited under AmericanType Culture Collection Accession Number ATCC ______ (referred to hereinas B2.6.2).
 11. A monoclonal antibody produced by the hybridoma cell ofclaim
 9. 12. A monoclonal antibody produced by the hybridoma cell ofclaim
 10. 13. A monoclonal antibody of claim 2 which binds tosubstantially the same epitope as the monoclonal antibody of claim 11.14. (Amended) A pharmaceutical composition comprising [a therapeuticallyeffective amount of] an antibody of claim 2 and a pharmaceuticallyacceptable carrier.
 15. An immunoassay comprising the steps of: (a)contacting a test sample with a monoclonal antibody of claim 1, and (b)determining the amount of hVEGF or hVEGF-related protein in the testsample that is bound to the monoclonal antibody.
 16. A monoclonalantibody of claim 2 which binds to substantially the same epitope as themonoclonal antibody of claim 12.